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BackgroundPancreatoduodenectomy for pancreatic head and periampullary cancers is still associated with high perioperative morbidity and mortality. The aim of this study was to compare the short-term outcomes of robot-assisted pancreatoduodenectomy (RAPD) and open pancreatoduodenectomy (OPD) performed in a high-volume centre.MethodsA single-centre, prospective database was used to retrospectively compare the early outcomes of RAPD procedures to standard OPD procedures completed between January 2014 and December 2018. Of the 121 included patients, 78 underwent RAPD and 43 underwent OPD. After propensity score matching (PSM), 35 RAPD patients were matched with 35 OPD patients with similar preoperative characteristics.ResultsThere were no statistically significant differences in most of the baseline demographics and perioperative outcomes in the two groups after PSM optimization with the exception of the operative time (530 min (RAPD) versus 335 min (OPD) post-match, p < 0.000). No differences were found between the two groups in terms of complications (including pancreatic leaks, 11.4% in both OPD and RAPD), perioperative mortality, reoperations or readmissions. Earlier refeeding was obtained in the RAPD group vs. the OPD group (3 vs. 4 days, p = 0.002). Although the differences in the length of the hospital stay and blood transfusions were not statistically significant, both parameters showed a positive trend in favour of RAPD. The number of harvested lymph nodes was similar and oncologically adequate.ConclusionsRAPD is a safe and oncologically adequate technique to treat malignancies arising from the pancreatic head and periampullary region. Several perioperative parameters resulted in trends favouring RAPD over OPD, at the price of longer operating time. Data should be reinforced with a larger sample to guarantee statistical significance.

In the Abstract, in the Methods section the sentence “Of the 121 included patients, 78 underwent RAPD and 43 underwent OPD.” Should read: Of the 121 included patients, 77 underwent OPD and 44 underwent RAPD.”

Compared to standardized minimally invasive colorectal procedures, there is considerable perioperative heterogeneity in loop ileostomy reversal. This study aimed to investigate the current perioperative practice and technical variations of loop ileostomy reversal following rectal cancer surgery. A nationwide online survey was conducted among members of the Italian Society of ColoRectal Surgery (SICCR). A link to the questionnaire was sent via mail. The survey consisted of 31 questions concerning the main procedural steps and application of the ERAS protocol after loop ileostomy reversal. Overall, 219 participants completed the survey. One respondent in four used a combination of water-soluble contrast studies (WSCS) and digital rectal examination to assess the integrity of the anastomosis before ileostomy closure. Conversely, 17.8% of them used either only WSCS or only endoscopy. Surgeons routinely perform hand-sewn or stapled anastomoses in 45.2% and 54.8% of the cases, respectively. Side-to-side antiperistaltic stapled anastomosis was the most performed anastomosis (36%). Most surgeons declared that they have never used prostheses for abdominal wall closure (64%), whereas 35% preferred retromuscular mesh placement in selected cases only. Forty-six respondents (66.7%) reported using interrupted stitches for skin closure, while 65 (29.7%) a purse-string suture. Furthermore, skin approximation at the stoma site using open methods was significantly more common among surgeons with greater experience in ileostomy reversal ( p  = 0.031). Overall, a good compliance with the ERAS protocol was found. However, colorectal surgeons were significantly more likely to follow the ERAS pathway than general surgeons ( p  < 0.05). Surgeons use different anastomotic techniques for ileostomy reversal after rectal cancer surgery. Based on current evidence, purse-string skin closure and ERAS pathway should be implemented, while the role of mesh prophylactic strategy needs to be explored further.

To date, gene therapy has employed viral vectors to deliver therapeutic genes. However, recent progress in molecular and cell biology has revolutionized the field of stem cells and gene therapy. A few years ago, clinical trials started using stem cell replacement therapy, and the induced pluripotent stem cells (iPSCs) technology combined with CRISPR-Cas9 gene editing has launched a new era in gene therapy for the treatment of neurological disorders. Here, we summarize the latest findings in this research field and discuss their clinical applications, emphasizing the relevance of recent studies in the development of innovative stem cell and gene editing therapeutic approaches. Even though tumorigenicity and immunogenicity are existing hurdles, we report how recent progress has tackled them, making engineered stem cell transplantation therapy a realistic option.

While it is accepted that extracellular vesicles (EVs)-mediated transfer of microRNAs contributes to intercellular communication, the knowledge about molecular mechanisms controlling the selective and dynamic miRNA-loading in EVs is still limited to few specific RNA-binding proteins interacting with sequence determinants. Moreover, although mutagenesis analysis demonstrated the presence/function of specific intracellular retention motifs, the interacting protein/s remained unknown. Here, PCBP2 was identified as a direct interactor of an intracellular retention motif: CLIP coupled to RNA pull-down and proteomic analysis demonstrated that it binds to miRNAs embedding this motif and mutagenesis proved the binding specificity. Notably, PCBP2 binding requires SYNCRIP, a previously characterized miRNA EV-loader as indicated by SYNCRIP knock-down. SYNCRIP and PCBP2 may contemporarily bind to miRNAs as demonstrated by EMSA assays and PCBP2 knock-down causes EV loading of intracellular microRNAs. This evidence highlights that multiple proteins/miRNA interactions govern miRNA compartmentalization and identifies PCBP2 as a dominant inhibitor of SYNCRIP function in murine hepatocytes.

PurposeTo assess the neurocognitive function and neurological toxicity of frameless linear accelerator (LINAC)-based stereotactic radiosurgery (SRS) in patients with 10 or more brain metastases (BM).Patients and methodsForty consecutive adult patients who received SRS for ten or more 10 BM < 3 cm in maximum size were evaluated. All plans were generated using a single-isocenter multiple-target (SIMT) SRS technique with doses of 22 Gy for lesions < 2 cm and 16–18 Gy for those ≥ 2 cm in size. Survival analyses were estimated by Kaplan–Meier method from the date of SRS. Neurocognitive function using the Hopkins verbal learning test-revised (HVLT-R) and activity of daily living scale (ADLS) were collected prospectively at baseline and at 3,6 and 12-month follow-up. Toxicity was assessed by the National Cancer Institute Common Toxicity Criteria for Adverse Events (Version 5.0).ResultsWith a median follow-up of 10.8 months, 1-year survival and local control rates were 65% and 86%, respectively. Grade 2 or 3 toxicity occurred in eleven patients, being associated with radiological changes suggestive of radiation necrosis in seven patients. Three months after SRS, the mean relative decline was 14.2% for HVLT-R delayed recall, 12.3% for HVLT-R recognition, and 9.8% for HVLT-R total recall. A significant deterioration of HVLT-R scores ranged from 5.5 to 18.7% of patients at different time points. ADLS scores declined over time, but changes were not significant.ConclusionsSRS is an effective and safe approach for patients with 10 or more BM able to maintain the pretreatment neurocognitive function in the majority of patients.

Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults, poses a formidable therapeutic challenge, due to its intrinsic radio- and chemoresistance and its ability to create a hostile, immunosuppressive tumor microenvironment (TME). Drug repurposing has emerged as a promising strategy to fight GBM. In this context, our efforts focused on chlorpromazine (CPZ), a first-generation antipsychotic agent previously shown by us to exert anti-tumor effects in both preclinical and clinical settings. We investigated the role of CPZ in remodeling the GBM microenvironment and shaping immune responses using four GBM cell lines, two standard anchorage-dependent models and two patient-derived neurospheres, enriched for tumoral stem cells. We determined cGAS-STING pathway activation and downstream gene expression via flow cytometry and RT-PCR. The cellular secretome following drug treatment was profiled via a luminescence cytokinome assay using a panel of 27 chemokines. Macrophages were phenotyped by flow cytometry using M1 and/or M2 specific markers and, finally, PD-L1 expression was assessed by quantitative flow cytometry and immunoblot analysis. We demonstrate that CPZ, alone or in combination with temozolomide (TMZ), the current standard of care, activates the cGAS-STING signaling pathway, thus promoting anti-tumor immune responses. Importantly, CPZ counteracts the immunosuppressive effects of TMZ, hindering some TMZ-induced processes as: i) induction of tumorigenic cytokines; ii) macrophage polarization toward a tumor-supportive M2-like phenotype, and iii) increase of PD-L1 expression, a key mechanism of immune evasion. This study uncovers that CPZ exerts a previously unrecognized anti-cancer immunomodulatory activity, remodeling the immune microenvironment and enhancing the anti-tumor immune response. By overcoming TMZ resistance, CPZ not only exerts a direct anti-neoplastic effect, but also sensitizes GBM cells to standard therapy.

Background The oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells. Methods Envisioning clinical application of the best characterized anti-HPV16 E6 and –HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed. Results We showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway. Conclusion Based on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

Background Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). Objective The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. Methods Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. Results RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14–34 and 31–50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. Conclusions Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.

Background HPV16 is implicated in around 30% of oropharyngeal cancers (OPCs). HPV-associated OPCs generally show a better prognosis, but 20% deviate from this trend, indicating a need for better molecular profiling. HPV16-E5 is an oncoprotein encoded by an mRNA that undergoes extensive splicing, with only one specific transcript being translatable. The prognostic significance of this E5-productive transcript in HPV-related OPCs is not well-studied. Methods We retrospectively analysed 74 HPV16-positive OPC cases diagnosed between 2011 and 2021. FFPE tissue samples were used for p16, EGFR, and HLA-I analysis by immunohistochemistry while E5, productive E5, E6 and E7 transcripts were detected by qPCR. Survival analysis was conducted using Kaplan-Meier curves and log-rank tests. Results Productive HPV16-E5 transcripts were detected in 11.3% of OPCs. There was no significant association between HPV16-E5 transcripts and EGFR or HLA-I expression. However, the presence of the productive E5 transcript was associated with worse progression-free survival (PFS) ( p  = 0.0024). EGFR or HLA-I expression was not significantly associated with PFS ( p  = 0.17 and p  = 0.93, respectively). Conclusions This study demonstrates for the first time the presence of E5-productive transcripts in HPV16-positive OPCs and its association with poorer PFS, highlighting its potential as a prognostic marker. Further research with larger cohorts is needed to validate these findings.